Clostridium difficile is the most common agent causing hospital-acquired diarrhea and colitis. It is now established that the peptide substance P (SP) is a critical mediator of the intestinal secretion and inflammation mediated by C. difficile toxin A. Two cell types that participate in the acute secretory and inflammatory responses to this toxin are the intestinal epithelial cells and intestinal macrophages. Functional substance P receptors (SPR) are markedly increased in both these cells within minutes of Clostridium difficile toxin A exposure and before secretory and inflammatory changes to this toxin are evident. Moreover, SPR in intestinal macrophages are functionally linked to the release of the proinflammatory cytokine TNFalpha and SP itself. The overall goal of this renewal application is to study the function and regulation of SPR in both these cells types. In aim 1 we will study the mechanism(s) of SPR upregulation on purified intestinal epithelial cells during toxin A enteritis and determine the effects of inflammatory mediators and substance P itself on expression of these receptors. The signal transduction pathways that lead to expression of substance P receptors in response to substance P will also be examined. In aim 2 we will examine whether proinflammatory cytokines can stimulate release of substance P from dorsal root, myenteric, and submucosal ganglia. In aim 3 we will examine if inflammatory cytokines are involved in increased expression of SP binding sites in purified intestinal macrophages and determine the mechanism of this upregulation. In aim 4 we will determine the pathophysiologic importance of SP receptors in activation of mast cells and intestinal macrophages in vivo using SP (NK-1) receptor deficient mice. All these studies will help understand the mechanism of SP receptor regulation during C. difficile toxin A- mediated acute intestinal secretion and inflammation and may lead to novel approaches for the treatment of intestinal inflammation.